The CGG repeat expansion in the 5′-UTR of the fragile X mental retardation gene (FMR1) has been implicated in the pathogenesis of two distinct disorders, fragile X syndrome (FXS), a neurodevelopmental disorder (1, 2), and fragile X-associated tremor and ataxia syndrome (FXTAS), a progressive neurodegenerative disease (3, 4). In the general population, the FMR1 5′-UTR contains 5 to 54 CGG repeats, while expansions of this trinucleotide repeat outside of the normal range fall into two distinct categories, the premutation range with 55 to 200 repeats and the full mutation range with greater than 200 and up to thousands of repeats (3, 5).
Full mutation expansions coupled with cytosine methylation result in transcriptional silencing of the FMR1 gene, loss of expression of FMR1 protein (FMRP), and FXS, the most common form of heritable X-linked mental retardation. The FXS phenotype is complex and highly variable, with mental impairment ranging from mild learning disabilities and emotional problems to severe mental retardation (1, 2). The severity of mental retardation is correlated with the degree of cytosine methylation of the FMR1 promoter and repeat region (6, 7).
Premutation alleles are associated with FXTAS, one of the most common single-gene forms of gait ataxia and tremor in older males (3, 4, 8, 9). While the full mutation alleles are transcriptionally silent, premutation alleles demonstrate a 2-10 fold increase in FMR1 mRNA levels (10) but normal or reduced amounts of FMRP(11-13). Since FXTAS is restricted to premutation carriers and not found in full mutation individuals, increased FMR1 transcript level rather than the reduced FMRP is thought to be the underlying cause of this disorder. Accordingly, post-mortem examination of the brains of FXTAS individuals revealed intranuclear inclusions containing the FMR1 transcript, ubiquitin, molecular chaperones, and components of the proteasome (14, 15). The current molecular model for FXTAS is that the transcript containing the expanded premutation size CGG repeat sequesters results in misfolding of cellular proteins, such as CGG-binding proteins, leading to the formation of intranuclear inclusions (16, 17). This model is consistent with the RNA gain-of-function model proposed for another trinucleotide-repeat associated disease, myotonic dystrophy (DM1), in which the CUG repeat in the 3′-UTR of DMPK sequesters RNA-binding proteins, such as the members of the muscleblind family (18, 19).
Although deregulation or mutations of FMR1 have been implicated in the pathogenesis of both FXS and FXTAS (1, 3, 20, 21), it is important to note that there have been individuals with phenotypic manifestation of FXS with no mutations found in the FMR1 gene (22, 23). In addition, only one-third of male premutation carriers develop FXTAS (9). These observations together with the wide variability of FXS and FXTAS phenotypes suggest that in addition to FMR1, there are other genes potentially involved in pathogenesis of these disorders.
The present invention provides the identification of a gene, ASFMR1, overlapping the CGG repeat region of the FMR1 gene in the antisense orientation. The ASFMR1 transcript is spliced, polyadenylated and exported to the cytoplasm. Similar to FMR1, the ASFMR1 transcript is elevated in lymphoblastoid cells and peripheral blood leukocytes of individuals with premutation alleles relative to normal and is not expressed from full mutation alleles. In addition, the ASFMR1 transcript exhibits premutation-specific alternative splicing, providing a qualitative molecular abnormality associated with FXTAS.
The present invention overcomes previous shortcomings in the art by providing methods and compositions for identifying subjects at increased risk of developing fragile X-associated tremor and ataxia syndrome (FXTAS) and identifying subjects at increased risk of developing fragile X syndrome (FXS).